P1217: TARGETING HSP110 IN COMBINATION WITH SELINEXOR IN PRIMARY MEDIASTINAL B-CELL LYMPHOMA AND IN CLASSICAL HODGKIN LYMPHOMA INHIBITS STAT6 ACTIVATION AND IMPAIRS LYMPHOMA CELL GROWTH
Authors: Manon Durand, Vincent Cabaud Gibouin, Vincent Camus, Leila Salmi, Laurence Duplomb, Pierre-Julien Viailly, Fabrice Jardin, Brigitte Sola, Carmen Garrido, Gaetan Jego
Journal: HemaSphere (2023)

Topic: 20. Lymphoma Biology & Translational Research Background: XPO1 is the major nuclear export protein for which the gene presents a recurrent mutation (XPO1E571K) in almost 25% of patients with primary mediastinal B-cell lymphoma (PMBL) or classical Hodgkin lymphoma (cHL). XPO1E571K alters the nucleocytoplasmic distribution of hundreds of proteins, thus promoting oncogenesis. Therefore, XPO1 is a key target in PBML and cHL. HSP110, a high molecular weight heat shock protein, is highly expressed in B cell lymphomas. It acts as a molecular chaperone for overexpressed or mutated proteins. HSP110 sustains permanent activation of survival and proliferation signalling pathways in cancer cells. Aims: Investigating the role of HSP110 towards proteins of the major survival pathways in the PMBL and cHL cell. First, we characterize the relationship between HSP110 and XPO1. Second, we determine the role of HSP110 in survival and proliferation of PMBL and cHL Methods: PMBL and cHL cell lines were Karpas-1106P, MedB-1, U-2940, L-428, L-1236, U-HO1. To demonstrate the interaction between proteins, we performed co-immunoprecipitations (co-IP), proximity ligation assays (PLA) and an in vitro thermophoresis experiment. To determine the impact of HSP110 on survival and proliferation, cells were treated with HSP110 inhibitors, transfected with siRNA or transfected with HSP110-GFP plasmid. In vivo experiments were performed in chicken embryo chorioallantoic membranes (CAM). Xenografts were treated with XPO1 inhibitor (0.1µM) and HSP110 inhibitor (5µM). Results: We observed by PLA a proximity of XPO1 and HSP110 in PMBL and cHL cell lines. Co-IP showed that HSP110 interacts with XPO1, either XPO1E571K or XPO1WT. HSP110 inhibition did not alter XPO1 expression, suggesting that HSP110 is not a molecular chaperone of XPO1. In contrast, XPO1 inhibition by selinexor, increased the nuclear localization of HSP110, suggesting that HSP110 is a cargo of XPO1. To determine the role of HSP110 in PMBL, we evaluated the overall survival (OS) and progression free survival (PFS) of PMBL patients based on the hsp110 gene expression. We observed a significantly higher OS and PFS in patients with low HSP110 mRNA expression. Using RNA interference, we demonstrated the dependency of PMBL and cHL cell lines towards HSP110 for survival and proliferation. PLA and co-IP showed that HSP110 interacts with XPO1 and STAT6. PMBL and cHL tumour progression in CAM xenografts was reduced by treatment with HSP110 inhibitor. Combining the HSP110 inhibitor with selinexor synergistically reduced tumour growth in vitro and in vivo. We observed a reduced activation of STAT6 after selinexor treatment in PMBL cell lines and in HEK-STAT6 IL-4-stimulated, that was amplified by the addition of HSP110 inhibitor. HSP110 inhibitor alone was also able to reduce STAT6 phosphorylation in the presence of IL-4 activation. We showed direct interaction of HSP110 and STAT6 by PLA, co-IP, and in vitro thermophoresis experiments. Treatment with an HSP110 inhibitor reduced the interaction of STAT6 with HSP110 in vitro and in vivo. Finally, we observed less degradation of STAT6 when HSP110 was overexpressed in the presence of the protein biosynthesis inhibitor cycloheximide. Summary/Conclusion: HSP110 is an essential protein for PMBL and cHL cell survival, through its interaction with STAT6 and its requirement for STAT6 phosphorylation. HSP110 is a cargo protein of XPO1 which is also required for STAT6 phosphorylation. HSP110 inhibitor and selinexor have a synergistic effect in reducing tumour growth, suggesting that this combination could be a promising new therapeutic strategy for PMBL and cHL. Keywords: Targeted therapy, Hodgkin’s lymphoma, Diffuse large B cell lymphoma, Heat shock protein